Chromatographic separation techniques are widely employed in the analysis of chemical components in complex samples. Because of their mechanical and chemical characteristics and because of their relatively low cost, silica surfaces are widely used to carry stationary phases in chromatographic processes. Most of the chromatographic stationary phases in use today are based on chemical modification of silica surfaces (Unger, ed., Packings and Stationary Phases in Chromatographic Techniques, New York:Marcel Dekker (1990) ("Unger")). Organic molecules with specific properties are chemically bonded to the silica surface to produce chromatographic stationary phases with desired functionality. This approach is based on the reaction of organosilanes with the silanol groups at the surface of the silica materials. However, these preparative methods require the use of highly reactive and corrosive silanization agents, which, in view of environmental concerns, are becoming increasingly impractical for the individual practitioner. The reactions are also very time consuming and need to be conducted in a controlled, inert environment, typically in a glove box or other such device. Furthermore, stationary phases formed with silanizing agents are chemically unstable, especially at high or low pH, which results in shortened life of the stationary phase and poor chromatographic separation.
In particular, open tubular liquid chromatography ("OTLC") and open tubular electrochromatography ("OTEC") have shown a great potential to reach high efficiencies for the analysis of complex sample mixtures (Dorsey et al., Anal. Chem., 66:531R (1994) ("Dorsey"), Ishii et al., J. Chromatogr. Sci., 18:462 (1980) ("Ishii"), and Kennedy et al., Science 246:57 (1989) ("Kennedy")). Preparation of the stationary phase is of key importance to achieve the high performance offered by these techniques.
However, capillary columns, especially capillary columns having less than about a 15 .mu.m inside diameter, coated with an appropriate stationary phase and having sufficient retentive and mass loadability characteristics, have been particularly difficult to prepare. This is one of the reasons why OTLC, for example, is presently being used in only a few research laboratories (Bruin et al. J. Chromatogr., 517:557 (1990)) despite its many advantages (Dorsey, Ishii, and Kennedy) over the more conventional high-performance liquid chromatography ("HPLC").
Although in the past 15 years efforts have been made to prepare stationary phases for capillary columns (Tsuda et al., J. Chromatogr., 11:332 (1978) ("Tsuda"), Jorgenson et al., J. Chromatogr., 255:335 (1988) ("Jorgenson"), Tock et al., J. Chromatogr., 477:95 (1989), Crego et al., Anal. Chem., 65:1615 (1993) ("Crego"), Dluzneski et al., J. High Resolut. Chromatogr. Chromatogr. Commun. 11:332 (1985) ("Dluzneski"), Folestad et al., J. Chromatogr., 391:347 (1987) ("Folestad"), and Bohlin et al., J. Chromatogr., 645:41 (1993) ("Bohlin")), there are still several problems associated with the methods developed so far.
Stationary phases that are chemically bonded directly to the inner wall of capillary columns offer very low phase ratios, which lead to low retention and low sample capacity (Tsuda and Jorgenson).
Methods developed to increase surface area and phase ratio involve two major steps: (1) laying down a porous silica layer and (2) attaching functional groups onto the prepared layer through chemical bonding (Tock et al., J. Chromatogr., 477:95 (1989) and Crego). Aside from the fact that these procedures are time consuming and require the manipulation of highly reactive and corrosive silanizing agents, the procedures are unreliable, frequently producing a significant number of columns which are unsuitable for use. For example, Crego reports success rates of less than 80%. Furthermore, the functional groups serving as the stationary phase are attached to the inner wall of the capillary columns (with or without the porous layer) through surface --O--Si--C bonds. These bonds are unstable at pH extremes, and this instability limits the pH range at which the columns can be operated.
Other methods for increasing surface area and phase ratio of capillary columns have employed polymeric stationary phases, such as cross-linked polysiloxanes, described in Dluzneski, Folestad, and Bohlin, and polyacrylates, disclosed in Ruan et al., Chromatographia, 35:597 (1993) and Swart et al., J. Chromatogr., 670:25 (1994). Although these stationary phases usually give good column stability, high phase ratio, and sufficient retention, they exhibit poor column efficiency, due to the slow diffusivity of solutes in the retentive layers.
The present invention is directed to overcoming these deficiencies in the art.